The gut is an important site of HIV acquisition. The majority of new HIV infections in the United States are acquired rectally, and rectal transmission of HIV is a significant mode of HIV acquisition globally. The gut is also a significant site of virs replication, CD4+ T cell depletion, inflammation and microbial translocation. Commensal gut microbiota protect the gut from infection by pathogenic organisms, however, gut microbiota can facilitate the transmission and pathogenesis of certain viruses that target the gut (i.e. poliovirus, norovirus, reovirus, mouse mammary tumor virus). The effect of gut microbiota on HIV acquisition risk and other aspects of HIV infection is not known. Our long-term goal is to establish how gut microbiota affect HIV acquisition, pathogenesis, latency, treatment and prevention. The direct experimentation that is needed to establish the role of human gut microbiota in HIV-1 acquisition and infection is not possible to perform in humans. Bone marrow/liver/thymus (BLT) humanized mice are systemically reconstituted with human immune cells and have been extensively utilized to study HIV transmission, pathogenesis and prevention strategies in vivo. However, the gut microbiome of BLT mice is murine. Recently, we rederived and utilized a germ-free immune deficient mouse strain to generate germ-free BLT mice that can be colonized with human gut microbiota (HuM-BLT mice). Our objective here is implement these novel and innovative in vivo models that we recently developed to evaluate the role of gut microbiota in rectal HIV-1 acquisition as the first step towards achieving this goal. Our preliminary data demonstrate: 1) robust systemic reconstitution of germ-free BLT mice with human HIV target cells; 2) the gut microbiome of HuM-BLT mice is stable and recapitulates the human donor inoculum; 3) HuM- BLT mice are susceptible to rectal HIV acquisition; and 4) the presence of human gut microbiota significantly increases rectal HIV acquisition in HuM-BLT mice in comparison to BLT mice with mouse microbiota (MM-BLT mice) (p=0.01). Based on our preliminary data, we hypothesize that the composition of the gut microbiome affects the rectal HIV-1 transmission risk. We will establish the full extent and utility of HuM-BLT mice for the study of the human gut microbiome and its role in rectal HIV-1 acquisition by generating multiple cohorts of HuM-BLT mice colonized with the gut microbiome of different healthy human donors and by 1) assessing the effect of human microbial diversity on gut microbiota colonization in HuM-BLT mice, 2) determining the effect of human gut microbiota on systemic reconstitution, maturation and activation of HIV-susceptible cells in HuM- BLT mice and 3) evaluating the role of gut microbiota in rectal HIV-1 acquisition, replication and pathogenesis. The information obtained will fill an important void in our knowledge regarding the role of human gut microbiota in rectal HIV-1 acquisition that in turn will serve to inform future clinical interventions aimed a preventing rectal HIV-1 transmission. These results will also validate HuM-BLT mice as an in vivo platform for the study of the human gut microbiome and for the evaluation of HIV infection in the context of human gut microbiota.